Separation And Identification Of Anthocyanins In Red Purple Tea Leaves

Anthocyanins are one of water-soluble pigments widely throughout the plant kingdom.They are classified to flavonoids and can attribute to antioxidant, anti-mutation activity and other biological functions. In this article ,the red-purple tea shoots were used as material, the analysis, isolation and purification method of anthocyanins in tea were investigated and several constituents were indentified. These works aim to provide the analysis method and therotic basis for studies of occurrence mechanism of red purple tea shoots, pharmacology and biological activity of anthocyanin monomer. The results are as follows:1. The HPLC analysis of anthocyanins in tea was established .The chromatogram separations were carried out on a BDS Hypersil C18 column. mobile solvents consisted of 2% formic acid aqueous solution(A)and acetonitrile (B), linear elution gradient from 10% to 40% B at 0 ~ 40min; Flow rate was 0.8 mL / min; injection volume was 10μL, column temperature was 28 o C, diode array detector was used and the detection wavelength was set at 520nm.2. Anathocyanins in crude tea extract was analyzed by using the established HPLC method and 9 peaks could be observed in the chromatogram , Among these peaks, only one peak contained two anathocyanin constituents, the others are purity; By using HPLC-DAD- MS/MS, the information including UV - visible spectrum , mass data of 7 types of tea anathocyanins were obtained. On the basis of above information and combined with the literature about sequence of anthocyanins in HPLC chromatogram, five main constituents of tea anthocyanins were identified as: delphinium -3 - galactoside, cyanidin -3 - galactosidase, delphinium -3 - rutinoside, cyanidin -3– rutinoside and geranium -3 - rutinoside. Otherwise, the molecular structure of two isomer constituents is necessary to be indentified in the future. 3. Using cyanidin -3 - glucosidase as the standard substance, the external quantity HPLC method of tea anthocyanins was established, the standard curve equation: Y = 1.87×107x-81360, R2 = 0.9985 . When the sample contained anthocyanins at the range of 0-0.30μg , the curve showed in good linear relationship. The purity of anthocyanins in crude tea extract was 32.0% by using the established HPLC method.4. The HSCCC method was applied to separate and purify anthocyanins in crude tea extract. The solvent system consisted of MTBE-n-butanol -acetonitrile-water- trifluoroacetic acid (3:1.5: 1:5:0.015 v / v). The upper layer was used as the stationary phase, the lower layer as the mobile phase; the instrument parameters for analytical HSCCC: revolution speed at 1800 r/min, flow rate at 0.8 mL / min, the temperature at 25℃, injection volume was 20μl; and for semi-preparative HSCCC : revolution speed at 900r/min, flow rate at 2mL/min, the temperature at 25℃, injection volume was 20mL.